Compositions for regenerating defective or absent myocardium

ABSTRACT

Compositions of the invention for regenerating defective or absent myocardium comprise an emulsified or injectable extracellular matrix composition. The composition can also have an extracellular matrix scaffold component of any formulation, then including also added cells, proteins, or other components to optimize the regenerative process and restore cardiac function. Methods for regenerating defective or absent myocardium apply a composition to a site of myocardium in need of regeneration using a delivery mode appropriate for the particular formulation.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a divisional of parent application Ser. No.11/182,551 filed Jul. 15, 2005.

FIELD OF THE INVENTION

The invention relates to tissue engineering generally, and morespecifically to compositions and methods for regenerating defective orabsent myocardium.

BACKGROUND OF THE INVENTION

Heart failure occurs in nearly 5 million people a year in the U.S. aloneat a combined cost of about $40 billion annually for hospitalization andtreatment of these patients. The results of all the effort and cost aredisappointing with a 75% five year mortality rate for the heart failurevictims. Treatments for chronic heart failure include medical managementwith pharmaceutical drugs, diet and exercise, transplantation for a fewlucky recipients, and mechanical assist devices, which are costly andrisk failure and infection. Thus the landscape for cardiac treatment isturning in recent years to transplantation of tissue or cells.

Medical researchers have transplanted human hematopoetic stem cells,mesenchymal stem cells, endothelial precursor cells, cardiac stem cells,and skeletal myoblasts or bone marrow cells to the myocardium, withhowever little or mixed success in satisfactory regeneration of themyocardium. Another protocol involved injecting transforming growthfactor beta preprogrammed bone marrow stem cells to the myocardium, withgreater success than transplantation of bone marrow stem cells alone,but without generation of contractile myocardium.

After myocardial infarction, injured cardiomyocytes are replaced byfibrotic tissue promoting the development of heart failure. On the basisthat embryonic stem cells may be directed to differentiate into truecardiomyocytes, transplantation of embryonic stem cells to a site ofmyocardial infarction may yield success in myocardial tissueregeneration, though the experiments have not yet so proven. For arelated challenge, to induce angiogenesis in ischemic myocardial tissue,transplanting endothelial progenitor cells, with or without angiogenicprotein factors has been proposed to generate capillary blood vessels atthe site of ischemia in the myocardium. As yet, the experiments to provethese theories have not worked sufficiently to be attempted in humans.

Meanwhile, typical structural abnormalities or damage to the heart thatwould lend itself to tissue regenerative therapies, were they available,include atrial septal defects, ventricular septal defects, rightventricular out flow stenosis, ventricular aneurysms, ventricularinfarcts, ischemia in the myocardium, infarcted myocardium, conductiondefects, conditions of aneurysmic myocardium, ruptured myocardium, andcongenitally defective myocardium, and these defective conditions remainuntreated in humans by any current tissue regenerative techniques.

Although tissue regeneration has been accomplished by transplantation inmammalian tissues such as the endocranium, the esophagus, blood vessels,lower urinary tract structures, and musculotendinous tissues, hearttissue regeneration by foreign tissue explant has remained a challenge.Recently, myocardium has been regenerated using xenogenic extracellularmatrix patches in pigs and dogs, and the contractility achieved was at90% of normal.

It would be beneficial for treatment of heart failure in humans todevelop myocardium regenerative strategies using matrices and additivesfor optimizing the potential results. One problem exists in thepreparation of extracellular scaffolds in that they must benon-immunogenic and thus acellular before implantation. Getting rid ofthe cells in the matrix may also inadvertently strip the scaffold of keybioactive proteins. In order to perform procedures to regenerate humanmyocardium with fidelity, compositions that mimic the function ofextracellular matrices are provided below.

No experimentation has been conducted to date on regenerating mammalianmyocardium using an emulsified or injectable extracellular matrixformulation. The only known experimental use of extracellular emulsionsfor tissue regeneration have been with gastroesophageal repair toprevent reflux and urinary bladder sphincter repair. Both of theseexperiments were conducted in non-human animals. Some veterinary use ofextracellular matrix emulsions have been reported, but none of thoseuses were for the repair of myocardium. The disadvantage of usingintact, non-emulsified extracellular matrix compositions such as patchesor strips is that placement of the material requires open surgery, withits coordinate risk of infection, challenge of access to the site, andlonger recovery for the patient post-procedure.

The present invention pioneers compositions and alternatives to priorart solutions for tissue regeneration to provide a biomedicalcomposition (and methods using the composition) for regeneratingdefective or absent myocardium, particularly for use in humans.

SUMMARY OF THE INVENTION

The invention contemplates a method of making a composition comprisingan extracellular matrix scaffold derived from a mammalian source and anadditional component selected from the groups consisting of:

a) a plurality of synthetic extracellular matrix-like scaffold-formingmolecules

b) a cell,

c) a peptide, polypeptide, or protein, and

d) a vector having a DNA capable of targeted expression of a selectedgene, and

e) a nutrient, a sugar, a fat, a lipid, an amino acid, a nucleic acid, aribo-nucleic acid, an organic molecule, an inorganic molecule, a smallmolecule, a drug, or a bioactive molecule,

said method comprising combining the extracellular matrix scaffold withone or more additional components, and storing the composition foradministration to a patient.

The scaffold is formulated in a material form selected from the groupconsisting of a solid sheet, multilaminate sheets, a gel, an emulsion,an injectable solution, a fluid, a paste, a powder, a plug, a strand, asuture, a coil, a cylinder, a weave, a strip, a spray, a vapor, a patch,a sponge, a cream, a coating, a lyophilized material, and avacuum-pressed material.

The additional component is formulated in a material form selected fromthe group consisting of a gel, an emulsion, an injectable solution, afluid, a paste, a powder, a plug, a strand, a spray, a vapor, a cream, acoating, a lyophilized material and a vacuum-pressed material.

The scaffold is derived from a source selected from the group consistingof small intestine submucosa (SIS), urinary bladder submucosa (UBS),urinary basement membrane (UBM), liver basement membrane (LBM), stomachsubmucosa (SS), collagen from animal sources, collagen from plantsources, synthesized extracellular matrix in cultures from cells(Matrigel™), dermal extracellular matrix material, subcutaneousextracellular matrix material, large intestine extracellular matrixmaterial, placental extracellular matrix material, ornamentumextracellular matrix material, heart extracellular matrix material, andlung extracellular matrix material.

The additional component is a plurality of synthetic extracellularmatrix-like scaffold-forming molecules, and said synthetic molecules areselected from the group consisting of polyethylene terephthalate fiber(Dacron), polytetrafluoroethylene (PTFE), glutaraldehyde-cross linkedpericardium, polylactate (PLA), polyglycol (PGA), hyaluronic acid,polyethylene glycol (PEG), polyethelene, a nucleic acid polymer,nitinol, and collagen from non-animal sources.

Further embodiments of the invention are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts cell-ECM interaction through the matrix proteoglycans,glycoaminoglycans and growth factors.

FIG. 2 depicts cell-cell adhesions, and cell-matrix adhesions throughspecific structural and functional molecules of the ECM.

FIG. 3 depicts a model of matrix scaffold structure including commoncollagen, proteoglycans, and glycoproteins.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The invention is a composition that regenerates defective or absentmyocardium and restores cardiac function. For this purpose, anemulsified or injectable extracellular matrix composition can be derivedfrom a mammalian or synthetic source. The composition can furtherinclude added cells or protein or both. An extracellular matrixcomposition of any formulation can include also an additional componentsuch as: a) a cell, b) a peptide, polypeptide, or protein, or c) avector expressing a DNA of a bioactive molecule, and d) other additiveslike nutrients or drug molecules. One additional component can be usedin the composition or several. The composition can be placed in contactwith the defective or absent myocardium, resulting in myocardial tissueregeneration and restoration of contractility, conductivity, or functionto the heart muscle. The invention appreciates the importance of thepresence of some amount and form of an extracellular matrix, orextracellular matrix-like scaffold, as a framework for the essentialactivities of cell-cell, matrix-cell, protein-cell, and protein-proteininteractions that form the dynamic tissue regenerative process in vivo,potentially optimized by the presence of added cells, proteins, or otherbioactive components.

Scaffold Requirements

A composition to accomplish regeneration of myocardium needs to inducecomplex dynamic interactions and activities at the site of defect. Thepresent invention provides a composition that creates an environment invivo to allow these processes to occur. The processes needed toregenerate myocardium include specific phenotypic changes in stem cellsthat are recruited to the defective site, establishment of cell-cellconnections, establishment of vascular supply at the site, beginning ofnormal tissue specific metabolism, limiting new growth once new tissueis made, coupling electric conduction from new cells to existing cellsand pathways, and establishment of cell-extracellular matrix connectionsby way of cell adhesions to the matrix proteins.

The expectations for the extracellular matrix scaffold are that it willorganize the cells into tissues, both by recruiting endogenous cells andusing cells that have been provided as additional components in thecomposition. The extracellular matrix scaffold then coordinates thefunction of the newly recruited or added cells, allowing also for cellmigration within the matrix. The matrix allows and provides for normalmetabolism to the cells once the vascular supply delivering nutrients tothe cells is established. Additionally, signal transduction pathways forgrowth, differentiation, proliferation and gene expression areestablished.

The extracellular matrix of myocardium is complex. There is athree-dimensional architecture established with proteoglycan molecules,with available cytokines in the microenvironment. Cell movement occursusing focal adhesions, and eventually permanent cell adhesions occurcalled hemidesmosomes. Environmental signals are transmitted, includingspecific cell signals from growth factors on cell surfaces and disposedwithin the matrix framework as well. The matrix itself has structuralcomponents and functional components and the line between the twosometimes blurs because some of the moieties of structural componentssignal and trigger protein activation, and activation of nearby cells.See FIG. 1 for an illustration of signaling, FIG. 2 for depiction ofcell-cell, protein-cell, and matrix-cell interactions, and FIG. 3 for adiagrammatic view of three-dimensional ECM scaffold.

There has been much research recently to elucidate the properties andfunction of the extracellular matrix: its protein make-up, and its rolein the body. The extracellular matrix (ECM) is a scaffold matrix ofpolymerized “structural” proteins that fit into three groups: collagens,glycoproteins, and proteoglycans (which have glycosaminoglycan repeatsthroughout). These molecules actually polymerize to form the scaffold ormatrix of proteins that exists in dynamic interaction with cells, andclosely placed functional proteins (either on the cells, or bound to astructural protein). Thus the extracellular matrix also includes withinits matrix scaffold “functional” proteins that interact with thestructural proteins and with migrating or recruited cells, particularlystem cells in tissue regeneration. The matrix functional proteins alsointeract with protein expressing cells during the life and maintenanceof the matrix scaffold itself as it rebuilds and maintains itscomponents. Note that some proteins fall into both a structural proteinclassification and a functional protein classification, depending on theprotein's configuration and placement in the whole matrix.

The extracellular matrix of myocardium is made up of collagen types I(which is predominant), III, IV, V, and VI, combined which are 92% ofthe dry weight of the matrix. Glycosaminoglycans (GAGs) includechondroitin sulfate A and B, heparan, heparin, and hyaluronic acid.Glycoproteins such as fibronectin and entactin, proteoglycans such asdecorin and perlecan, and growth factors such as transforming growthfactor beta (TGF-beta), fibroblast growth factor-2 (FGF-2) and vascularendothelial growth factor (VEGF), are key players in the activity of amyocardium regenerating matrix. Furthermore, the precise chemicalconstitution of the matrix appears to play a role in its function,including for example what collagen type is prevalent in the matrix, thepore size established by the matrix scaffold, the forces transmitted toadhesion molecules and mechanoreceptors in the cell membranes of cellsat the matrix, and the forces directed from the three-dimensionalenvironment (for example the gene expression in the three-dimensionalmatrix scaffold environment is very different than in a monolayerenvironment). Thus, the outcome of any tissue regenerative processes isdetermined by the structural and functional components of the matrixscaffold that form the basis of the regenerative process.

More specifically, when in early regenerative processes, circulatingcells or added cells are directed, initial temporary cell adhesionprocesses occur that result in embryogenesis of the cells, morphogenesisof the cells, regeneration of cell form, eventual maintenance of thecell, possible motility to another site, and organogenesis that furtherdifferentiates the cell. Facilitating these early cell adhesionfunctions are cell adhesion molecules (CAMs). The CAMs are availableeither endogenously, or added as an additional component of thecomposition. CAMs are glycoproteins lodged in the surface of the cellmembrane or transmembrane connected to cytoskeletal components of thecell. Specific CAMs include cadherins that are calcium dependent, andmore than 30 types are known. Also working as CAMs are integrins whichare proteins that link the cytoskeleton of the cell in which they arelodged to the extracellular matrix or to other cells through alpha andbeta transmembrane subunits on the integrin protein. See FIG. 2 for anillustration of these interactions. Cell migration, embryogenesis,hemostatis, and wound healing are so facilitated by the integrins in thematrix. Syndecans are proteoglycans that combine with ligands forinitiating cell motility and differentiation. Immunoglobins provide anynecessary immune and inflammatory responses. Selectins promote cell-cellinteractions.

Specific requirements for the scaffold component of the invention,whether a native scaffold prepared for introduction into a mammal, or asynthetic scaffold formed by synthetic polymerizing molecules, or acombination of the two, are that the scaffold must be resorbable overtime as the tissue regeneration ensues, and this resorbtion is at anappropriate degradation rate for optimal tissue regeneration and absenceof scar tissue formation. The extracellular matrix scaffold must also benon-toxic, provide a three-dimensional construction at the site ofdefect in the myocardium (once delivered to the site). The matrixscaffold is required to have a high surface area so that there is plentyof room for the biological activities required of the tissueregeneration process. The scaffold must be able to provide cellularsignals such as those mentioned herein that facilitate tissueregeneration. Finally the scaffold needs to be non-immunogenic so thatit is not rejected by the host, and it needs to be non-thrombogenic.

Particular study of the components of the native scaffolds facilitatesdesign of compositions well-suited for regeneration of myocardium.

The Structural Proteins of the Extracellular Matrix Scaffold

Collagens, the most abundant components of ECM, are homo- orheterotrimeric molecules whose subunits, the alpha chains, are distinctgene products. To date 34 different alpha chains have been identified.The sequence of the alpha chains contains a variable number of classicalGly-X-Y repetitive motifs which form the collagenous domains andnoncollagenous domains. The collagenous portions of 3 homologous orheterologous alpha chains are folded together into a helix with a coiledcoil conformation that constitutes the basic structure motif ofcollagens.

Characteristically, collagens form highly organized polymers. Two mainclasses of molecules are formed by collagen polymers: the fibril-formingcollagens (collagens type I, II, III, V, and XI) and the non-fibrillarcollagens that are a more heterogeneous class. Fibril collagen moleculesusually have a single collagenous domain repeated the entire length ofthe molecule, and non-fibrillar collagen molecules have a mixture ofcollagenous and noncollagenous domains. On this basis several moresubgroups of the collagen family are identified: e.g. the basementmembrane collagens (IV, VIII, and X). In addition, most all thedifferent types of collagen have a specific distribution. For example,fibril forming collagens are expressed in the interstitial connectivetissue. The most abundant component of basement membranes is collagenIV. The multiplexins, collagens XV and XVIII are also localized to thebasement membranes.

In the extracellular matrix of the heart, collagen types I and IIIpredominate, together forming fibrils and providing most of theconnective material for typing together myocytes and other structures inthe myocardium, and thus these molecule types are involved in thetransmission of developed mechanical force in the heart. Only collagentypes I, II, III, V, and XI self assemble into fibrils, characterized bya triple helix in the collagen molecules. Some collagens form networks,as with the basement membrane, formed by collagen IV. Type III collagendominates in the wall of blood vessels and hollow intestinal organs andcopolymerizes with type I collagen.

Proteoglycans are grouped into several families, and all have a proteincore rich in glycosoaminoglycans. They control proliferation,differentiation, and motility. The lecticans interact with hyaluronanand include aggrecan, versican, neurocan, and brevican. Versicanstimulates proliferation of fibroblasts and chondrocytes through thepresence in the molecule of EGF-like motifs. The second type ofproteoglycans have a protein core with leucine-rich repeats, which forma horse shaped protein good for protein-protein interactions. Theirglycosoaminoglycan side chains are mostly chondroitin/dermatan sulphateor keratin sulphate. Decorin, biglycan, fibromodulin, and keratocan aremembers of this family. Decorin is involved in modulation anddifferentiation of epithelial and endothelial cells. In addition,transforming growth factor beta (TGF beta) interacts with members ofthis family. There are part-time proteoglycans, comprising CD44 (areceptor for hyaluronic acid), macrophage colony stimulating factor,amyloid precursor protein and several collagens (IX, XII, XIV, andXVIII). The last family of proteoglycans is the heparan sulfateproteoglycans, some of which are located in the matrix, and some ofwhich are on cell membranes. Perlecan and agrin are matrix heparansulfate proteoglycans found in basement membranes. The syndecans andglypicans are membrane-associated heparan sulfate proteoglycans.Syndecans have a heparan sulfate extracellular moiety that binds withhigh affinity cytokines and growth factors, including fibroblast growthfactor (FGF), hepatocyte growth factor (HGF), platelet-derived growthfactor (PDGF), heparin-binding epidermal growth factor (HB-EGF), andvascular endothelial growth factor (VEGF). The heparan sulfateproteoglycans have been implicated in modulation of cell migration,proliferation and differentiation in wound healing.

Glycoproteins are also structural proteins of ECM scaffold. Theglycoprotein fibronectin (Fn) is a large dimer that attracts stem cells,fibroblasts and endothelial cells to a site of newly forming matrix.Tenascin is a glycoprotein that has Fn repeats and appears during earlyembryogenesis then is switched off in mature tissue. Tenascin reappearsduring wound healing. Other glycoprotein components of ECM includeelastin that forms the elastic fibers and is a major structuralcomponent along with collagen; fibrillins which are a family of proteinsconsisting almost entirely of endothelial growth factor (EGF)-likedomains. Small glycoproteins present in ECM include nidogen/entactin andfibulins I and II.

The glycoprotein laminin is a large protein with three distinctpolypeptide chains. Together with type IV collagen, nidogen, andperlecan, laminin is one of the main components of the basementmembrane. Laminin isoforms are synthesized by a wide variety of cells ina tissue-specific manner. Laminin I contains multiple binding sites tocellular proteins. Virtually all epithelial cells synthesize laminin, asdo small, skeletal, and cardiac muscle, nerves, endothelial cells, bonemarrow cells, and neuroretina. Laminins affect nearby cells, bypromoting adhesion, cell migration, and cell differentiation. They exerttheir effects mostly through binding to integrins on cell surfaces.Laminins 5 and 10 occur predominantly in the vascular basement membraneand mediate adhesion of platelets, leukocytes, and endothelial cells.

The Functional Proteins of the Extracellular Matrix Scaffold

In addition to the structural matrix proteins just discussed, specificinteractions between cells and the ECM are mediated by functionalproteins of the ECM, including transmembrane molecules, mainlyintegrins, some members of the collagen family, some proteoglycans,glycosaminoglycan chains, and some cell-surface associated proteins.These interactions lead to direct or indirect control of cellularactivities within the extracellular matrix scaffold such as adhesion,migration, differentiation, proliferation, and apoptosis.

Glycosaminoglycans (GAGs) are glycosylated post-translational moleculesderived from proteoglycans. Well known GAGs include heparin, hyaluronicacid, heparan sulfate, and chondroitin sulfate A, B, and C. Heparinchains stimulate angiogenesis, and act as subunits in a proteoglycan tostimulate the angiogenic effects of fibroblast growth factor-2 (FGF-2)(also known as basic FGF or bFGF). Chondroitin sulfate B (dermatansulfate) interacts with TGF-beta to control matrix formation andremodeling. The proteoglycan form of chondroitin sulfate B regulates thestructure of ECM by controlling collagen fibril size, orientation anddeposition. Hyaluronic acid is associated with rapid wound healing andorganized deposit of collagen molecules in the matrix. It is believedthat hyaluronic acid binds TGF-beta1 to inhibit scar formation.

The ECM is also being remodeled constantly in the live animal. Theproteins of the ECM are broken down by matrix metalloproteases, and newprotein is made and deposited as replacement protein. Collagens aremostly synthesized by the cells comprising the ECM: fibroblasts,myofibroblasts, osteoblasts, and chondrocytes. Some collagens are alsosynthesized by adjacent parenchymal cells or also covering cells such asepithelial, endothelial, or mesothelial cells.

The extracellular part of integrins bind fibronectin, collagen andlaminin, and act primarily as adhesion molecules. Integrin-ligandbinding also triggers cascades of activity for cell survival, cellproliferation, cell motility, and gene transcription.

Tenascins include cytotactin (TN-C). Cell surface receptors fortenascins include integrins, cell adhesion molecules of the Igsuperfamily, a transmembrane chrondroitin sulfate proteoglycan(phosphacan) and annexin II. TN-C also interacts with extracellularproteins such as fibronectin and the lecticans (the class ofextracellular chondroitin sulphate proteoglycans including aggrecan,versican, and brevican).

In addition to direct knowledge of protein cell interaction many of theproteins associated with the ECM can initiate binding to proteins thatthen activate to bind other proteins or cells, e.g. decorin binds Fn orthrombospondin and causes their cell adhesion promoting activity. Otherproteoglycans control the hydration of the ECM and the spacing betweenthe collagen fibrils and network, which is believed to facilitate cellmigration. Proteoglycans regulate cell function by controlling growthfactor activity, e.g. decorin, biglycan, and fibromodulin bind toisoforms of transforming growth factor beta (TGF beta) and heparinsulfate proteoglycans bind and store fibroblast growth factor.

The matrix metalloproteases (MMPs) break down the collagen molecules inthe ECM so that new collagen can be used to remodel and renew the ECMscaffold. It is also believed that the proteolytic activity of MMPsaugment the bioavailability of growth factors sequestered within theECM, and can activate latent secreted growth factors like TGF-beta andIGF from IGFBPs and cell surface growth factor precursors. MMPs canproteolytically cleave cell surface growth factors, cytokines, chemokinereceptors and adhesion receptors, and thus participate in controllingresponses to growth factors, cytokines, chemokines, as well as cell-celland cell-ECM interactions.

Structural or functional matrix proteins that can comprise thecompositions herein disclosed to facilitate myocardial tissueregeneration include, minimally, collagen I and III, elastin, laminin,CD44, hyaluronan, syndecan, bFGF, HGF, PDGF, VEGF, Fn, tenascin,heparin, heparan sulfate, chondroitin sulfate B, integrins, decorin, andTGF-beta.

Native Sources and Preparations

Native extracellular matrix scaffolds, and the proteins that form them,are found in their natural environment, the extracellular matrices ofmammals. These materials are prepared for use in mammals in tissuegrafts procedures. Small intestine submucosa (SIS) is described in U.S.Pat. No. 5,275,826, urinary bladder submucosa (UBS) is described in U.S.Pat. No. 5,554,389, stomach submucosa (SS) is described in U.S. Pat. No.6,099,567, and liver submucosa (LS) or liver basement membrane (LBM) isdescribed in U.S. Pat. No. 6,379,710, to name some of the extracellularmatrix scaffolds presently available for explanting procedures. Inaddition, collagen from mammalian sources can be retrieved from matrixcontaining tissues and used to form a matrix composition. Extracellularmatrices can be synthesized from cell cultures as in the productmanufactured by Matrigel™. In addition, dermal extracellular matrixmaterial, subcutaneous extracellular matrix material, large intestineextracellular matrix material, placental extracellular matrix material,ornamentum extracellular matrix material, heart extracellular matrixmaterial, and lung extracellular matrix material, may be used, derivedand preserved similarly as described herein for the SIS, SS, LBM, andUBM materials. Other organ tissue sources of basement membrane for usein accordance with this invention include spleen, lymph nodes, salivaryglands, prostate, pancreas and other secreting glands. In general, anytissue of a mammal that has an extracellular matrix can be used fordeveloping an extracellular matrix component of the invention.

When using collagen-based synthetic ECMs, the collagenous matrix can beselected from a variety of commercially available collagen matrices orcan be prepared from a wide variety of natural sources of collagen.Collagenous matrix for use in accordance with the present inventioncomprises highly conserved collagens, glycoproteins, proteoglycans, andglycosaminoglycans in their natural configuration and naturalconcentration. Collagens can be from animal sources, from plant sources,or from synthetic sources, all of which are available and standard inthe art.

The proportion of scaffold material in the composition when nativescaffold used will be large, as the natural balance of extracellularmatrix proteins in the native scaffolds usually represents greater than90% of the extracellular matrix material by dry weight. Accordingly, fora functional tissue regenerative product, the scaffold component of thecomposition by weight will be generally greater than 50% of the totaldry weight of the composition. Most typically, the scaffold willcomprise an amount of the composition by weight greater than 60%,greater than 70%, greater than 80%, greater than 82%, greater than 84%,greater than 86%, greater than 88%, greater than 90%, greater than 92%,greater than 94%, greater than 96%, and greater than 98% of the totalcomposition.

Native extracellular matrices are prepared with care that theirbioactivity for myocardial tissue regeneration is preserved to thegreatest extent possible. Key functions that may need to be preservedinclude control or initiation of cell adhesion, cell migration, celldifferentiation, cell proliferation, cell death (apoptosis), stimulationof angiogenesis, proteolytic activity, enzymatic activity, cellmotility, protein and cell modulation, activation of transcriptionalevents, provision for translation events, inhibition of somebioactivities, for example inhibition of coagulation, stem cellattraction, and chemotaxis. Assays for determining these activities arestandard in the art. For example, material analysis can be used toidentify the molecules present in the material composition. Also, invitro cell adhesion tests can be conducted to make sure that the fabricor composition is capable of cell adhesion.

The matrices are generally decellularized in order to render themnon-immunogenic. A critical aspect of the decellularization process isthat the process be completed with some of the key protein functionretained, either by replacement of proteins incidentally extracted withthe cells, or by adding exogenous cells to the matrix composition aftercell extraction, which cells produce or carry proteins needed for thefunction of tissue regeneration in vivo.

Myocardial tissue has been regenerated in vivo in non-humans usingnative xenogenic extracellular matrix scaffolds in the form of intactpatches derived and prepared from mammals, so it can be presumed that atleast some of the components required for myocardial tissue regenerationare to be found in these xenogenic patch matrices. Prudent practice maydictate that the cell extract from the patches be tested for its proteinmake-up, so that if necessary proteins are removed they can be placeback into the matrix composition, perhaps using exogenous proteins atapproximately the same amount as those detected in the extractionsolution. Replacing lost essential proteins may also be necessary withemulsions or injectable solutions of extracellular matrix, particularlythose emulsified from mammalian sources. Another option would be thatthe proteins extracted during the cell extraction process can simply beadded back after the cell extraction is complete, thus preserving thedesired bioactivity in the material.

The bioactivity of extracellular matrix material can be mimicked intissue regeneration experiments with combinations of native andsynthetic extracellular matrices explanted together, also optionallywith additional components such as proteins or cells, in order toprovide an optimal myocardial tissue regenerative composition andenvironment in vivo. What works as the best composition for myocardialtissue regeneration in patients, particularly humans can be tested firstin other mammals by standard explanting procedures to determine whethertissue regeneration is accomplished and optimized by a particularcomposition. See Badylak et al, The Heart Surgery Forum, ExtracellularMatrix for Mvocardial Repair 6(2) E20-E26 (2003).

When adding proteins to the extracellular matrix composition, be it anemulsified composition, or another formulation of matrix, the proteinsmay be simply added with the composition, or each protein may becovalently linked to a molecule in the matrix. Standard protein-moleculelinking procedures may be used to accomplish the covalent attachment.

For decellularization when starting with a whole organ, whole organperfusion process can be used. The organ is perfused with adecellularization agent, for example 0.1% peractic acid rendering theorgan acellular. The organ can then be cut into portions and stored(e.g. in aqueous environment, liguid nitrogen, cold, freeze-dried, orvacuum-pressed) for later use. Any appropriate decellularizing agent maybe used in whole organ perfusion process.

With regard to submucosal tissue, extractions may be carried out a nearneutral pH (in a range from about pH 5.5 to about pH 7.5) in order topreserve the presence of growth factor in the matrices. Alternatively,acidic conditions (i.e. less than 5.5 pH) can be used to preserved thepresence of glycosaminoglycan components, at a temperature in a rangebetween 0 and 50 degrees centrigrade. In order to regulate the acidic orbasic environment for these aqueous extractions, a buffer and chaotropicagent (generally at a concentration from about 2M to about 8M) areselected, such as urea (at a concentration from about 2M to 4M),guanidine (at a concentration from about 2M to about 6M, most typicallyabout 4M), sodium chloride, magnesium chloride, and non-ionic or ionicsurfactants. Urea at 2M in pH 7.4 provides extraction of basis FGF andthe glycoprotein fibronectin. Using 4M guanidine with pH 7.4 bufferyields a fraction having transforming growth factor beta. (TGF-beta).Accordingly, it may behoove a practitioner to decellularize one portionof a matrix, and extract desired proteins to add back in from otherdifferent portions.

Because of the collagenous structure of basement membrane and the desireto minimize degradation of the membrane structure during celldissociation, collagen specific enzyme activity should be minimized inthe enzyme solutions used in the cell-dissociation step. For example,liver tissue is typically also treated with a calcium chelating agent orchaotropic agent such as a mild detergent such as Triton 100. The celldissociation step can also be conducted using a calcium chelating agentor chaotropic agent in the absence of an enzymatic treatment of thetissue. The cell-dissociation step can be carried out by suspendingliver tissue slices in an agitated solution containing about 0.05 toabout 2%, more typically about 0.1 to about 1% by weight protease,optionally containing a chaotropic agent or a calcium chelating agent inan amount effective to optimize release and separation of cells from thebasement membrane without substantial degradation of the membranematrix.

After contacting the liver tissue with the cell-dissociation solutionfor a time sufficient to release all cells from the matrix, theresulting liver basement membrane is rinsed one or more times withsaline and optionally stored in a frozen hydrated state or a partiallydehydrated state until used as described below. The cell-dissociationstep may require several treatments with the cell-dissociation solutionto release substantially all cells from the basement membrane. The livertissue can be treated with a protease solution to remove the componentcells, and the resulting extracellular matrix material is furthertreated to remove or inhibit any residual enzyme activity. For example,the resulting basement membrane can be heated or treated with one ormore protease inhibitors.

Basement membrane or other native ECM scaffolds may be sterilized usingconventional sterilization techniques including tanning withglutaraldehyde, formaldehyde tanning at acidic pH, ethylene oxidetreatment, propylene oxide treatment, gas plasma sterilization, gammaradiation, and peracetic acid sterilization. A sterilization techniquewhich does not significantly weaken the mechanical strength andbiotropic properties of the material is preferably used. For instance,it is believed that strong gamma radiation may cause loss of strength inthe graft material. Preferred sterilization techniques include exposingthe graft to peracetic acid, low dose gamma irradiation and gas plasmasterilization; peracetic acid sterilization being the most preferredmethod.

Synthetics

Synthetic extracellular matrices can be formed using synthetic moleculesthat polymerize much like native collagen and which form a scaffoldenvironment that mimics the native environment of mammalianextracellular matrix scaffolds. According, such materials aspolyethylene terephthalate fiber (Dacron), polytetrafluoroethylene(PTFE), glutaraldehyde-cross linked pericardium, polylactate (PLA),polyglycol (PGA), hyaluronic acid, polyethylene glycol (PEG),polyethelene, nitinol, and collagen from non-animal sources (such asplants or synthetic collagens), can be used as components of a syntheticextracellular matrix scaffold. The synthetic materials listed arestandard in the art, and forming hydrogels and matrix-like materialswith them is also standard. Their effectiveness can be tested in vivo assited earlier, by testing in mammals, along with components thattypically constitute native ECMs, particularly the growth factors andcells responsive to them.

The ECM-like materials are described generally in the review article“From Cell-ECM Interactions to Tissue Engineering” Rosso et al, Journalof Cellular Physiology 199:174-180 (2004). In addition, some ECM-likematerials are listed here. Particularly useful biodegradable and/orbioabsorbable polymers include polylactides, poly-glycolides,polycarprolactone, polydioxane and their random and block copolymers.Examples of specific polymers include poly D,L-lactide,polylactide-co-glycolide (85:15) and polylactide-co-glycolide (75:25).Preferably, the biodegradable and/or bioabsorbable polymers used in thefibrous matrix of the present invention will have a molecular weight inthe range of about 1,000 to about 8,000,000 g/mole, more preferablyabout 4,000 to about 250,000 g/mole. The biodegradable and/orbioabsorbable fiberizable material is preferably a biodegradable andbioabsorbable polymer. Examples of suitable polymers can be found inBezwada, Rao S. et al. (1997) Poly(p-Dioxanone) and its copolymers, inHandbook of Biodegradable Polymers, A. J. Domb, J. Kost and D. M.Wiseman, editors, Hardwood Academic Publishers, The Netherlands, pp.29-61. The biodegradable and/or bioabsorbable polymer can contain amonomer selected from the group consisting of a glycolide, lactide,dioxanone, caprolactone, trimethylene carbonate, ethylene glycol andlysine. The material can be a random copolymer, block copolymer or blendof monomers, homopolymers, copolymers, and/or heteropolymers thatcontain these monomers. The biodegradable and/or bioabsorbable polymerscan contain bioabsorbable and biodegradable linear aliphatic polyesterssuch as polyglycolide (PGA) and its random copolymerpoly(glycolide-co-lactide-) (PGA-co-PLA). The FDA has approved thesepolymers for use in surgical applications, including medical sutures. Anadvantage of these synthetic absorbable materials is their degradabilityby simple hydrolysis of the ester backbone in aqueous environments, suchas body fluids. The degradation products are ultimately metabolized tocarbon dioxide and water or can be excreted via the kidney. Thesepolymers are very different from cellulose based materials, which cannotbe absorbed by the body.

Other examples of suitable biocompatible polymers are polyhydroxyalkylmethacrylates including ethylmethacrylate, and hydrogels such aspolyvinylpyrrolidone, polyacrylamides, etc. Other suitable bioabsorbablematerials are biopolymers which include collagen, gelatin, alginic acid,chitin, chitosan, fibrin, hyaluronic acid, dextran, polyamino acids,polylysine and copolymers of these materials. Any glycosaminoglycan(GAG) type polymer can be used. GAGs can include, e.g., heparin,chondroitin sulfate A or B, and hyaluronic acid, or their syntheticanalogues. Any combination, copolymer, polymer or blend thereof of theabove examples is contemplated for use according to the presentinvention. Such bioabsorbable materials may be prepared by knownmethods.

Nucleic acids from any source can be used as a polymeric biomaterial.Sources include naturally occurring nucleic acids as well as synthesizednucleic acids. Nucleic acids suitable for use in the present inventioninclude naturally occurring forms of nucleic acids, such as DNA(including the A, B and Z structures), RNA (including mRNA, tRNA, andrRNA together or separated) and cDNA, as well as any synthetic orartificial forms of polynucleotides. The nucleic acids used in thepresent invention may be modified in a variety of ways, including bycross linking, intra-chain modifications such as methylation andcapping, and by copolymerization. Additionally, other beneficialmolecules may be attached to the nucleic acid chains. The nucleic acidsmay have naturally occurring sequences or artificial sequences. Thesequence of the nucleic acid may be irrelevant for many aspects of thepresent invention. However, special sequences may be used to prevent anysignificant effects due to the information coding properties of nucleicacids, to elicit particular cellular responses or to govern the physicalstructure of the molecule. Nucleic acids may be used in a variety ofcrystalline structures both in finished biomaterials and during theirproduction processes. Nucleic acid crystalline structure may beinfluenced by salts used with the nucleic acid. For example, Na, K, Biand Ca salts of DNA all have different precipitation rates and differentcrystalline structures. Additionally, pH influences crystallinestructure of nucleic acids.

The physical properties of the nucleic acids may also be influenced bythe presence of other physical characteristics. For instance, inclusionof hairpin loops may result in more elastic biomaterials or may providespecific cleavage sites. The nucleic acid polymers and copolymersproduced may be used for a variety of tissue engineering applicationsincluding to increase tissue tensile strength, improve wound healing,speed up wound healing, as templates for tissue formation, to guidetissue formation, to stimulate nerve growth, to improve vascularizationin tissues, as a biodegradable adhesive, as device or implant coating,or to improve the function of a tissue or body part. The polymers mayalso more specifically be used as sutures, scaffolds and wounddressings. The type of nucleic acid polymer or copolymer used may affectthe resulting chemical and physical structure of the polymericbiomaterial.

Emulsions and Injectables

The extracellular matrix can be emulsified for administration to thedefective or absent myocardium. The matrix may also be otherwiseliquefied or made into an injectable solution, such as an emulsion, or aliquid, or injectable gel, or semi-gel, other injectable formulationthat can be administered with a percutaneous catheter, or other devicecapable of delivering an injectable formulation.

An emulsion of mammalian or synthetic extracellular matrix material canbe accomplished as is standard for tissue or polymer emulsification ingeneral. Generally, the emulsion will be maintained in an emulsifiedstate by control of some component of the composition, for example thepH. Upon delivery of the emulsion the pH is altered to allow themolecules of the matrix to polymerize into a three-dimensional scaffold.

An emulsified extracellular matrix material comprising also cells canhave the cultured cells simply added into the matrix emulsion, or thecells may be co-cultured with the matrix for a time beforeadministration to the patient. Standard procedures for culturing orco-culturing cells can be used.

In addition, where proteins such as growth factors, or any otherprotein, including protein forms such as peptides or polypeptides, orprotein fragments, are added into the extracellular matrix, the proteinmolecules may be added into the matrix composition, or the proteinmolecules may be covalently linked to a molecule in the matrix. Thecovalent linking of protein to matrix molecules can be accomplished bystandard covalent protein linking procedures known in the art. Theprotein may be covalently linked to one or more matrix molecules. Thecovalent linking may result in an integration of the protein moleculesin the matrix scaffold formation once the emulsion converts from theemulsified form to the scaffold form of the extracellular matrix.

Additional Components: Cells

Unlike skeletal myocytes, cardiomyocytes withdraw from cell cycleshortly after birth, and adult mammalian cardiomyocytes lack thepotential to proliferate. Therefore, in order to regenerate myocardium,the right cells may have to be added to the composition, or the site, orthe right molecules to attract the right cells will have to be added tothe composition or the site. Transplantation cell sources for themyocardium include allogenic, xenogenic, or autogenic sources.Accordingly, human embryonic stem cells, neonatal cardiomyocytes,myofibroblasts, mesenchymal cells, autotransplanted expandedcardiomyocytes, and adipocytes can be used as additive components toaccompany the scaffold.

Embryonic stem cells begin as totipotent cells, differentiate topluripotent cells, and then further specialization. They are cultured exvivo and in the culture dish environment differentiate either directlyto heart muscle cells, or to bone marrow cells that can become heartmuscle cells. The cultured cells are then transplanted into the mammal,either with the composition or in contact with the scaffold and othercomponents.

Myoblasts are another type of cell that lend themselves totransplantation into myocardium, however, they do not always developinto cardiomyocytes in vivo. Adult stem cells are yet another species ofcell that work in the context of tissue regeneration. Adult stem cellsare thought to work by generating other stem cells (for example thoseappropriate to myocardium) in a new site, or they differentiate directlyto a cardiomyocyte in vivo. They may also differentiate into otherlineages after introduction to organs, such as the heart. The adultmammal provides sources for adult stem cells in circulating endothelialprecursor cells, bone marrow-derived cells, adipose tissue, or cellsfrom a specific organ. It is known that mononuclear cells isolated frombone marrow aspirate differentiate into endothelial cells in vitro andare detected in newly formed blood vessels after intramuscularinjection. Thus, use of cells from bone marrow aspirate may yieldendothelial cells in vivo as a component of the composition.

Yet another viable option for cells to use in the invention are themesenchymal stem cells administered with activating cytokines.Subpopulations of mesenchymal cells have been shown to differentiatetoward myogenic cell lines when exposed to cytokines in vitro.

Once a type of cell is chosen, the number of cells needed is determined.Their function and anticipated change upon implantation, as well astheir viability during the process of transplantation need to beconsidered to determine the number of cells to transplant. Also the modeof transplantation is to be considered: several modes includingintracoronary, retrograde venous, transvascular injection, directplacement at the site, thoracoscopic injection and intravenous injectioncan be used to put the cells at the site or to incorporate them with thecomposition either before delivery or after delivery to the defectivemyocardium. In all cases, the mode of delivery and whether the cells arefirst mixed with the other components of the composition is a decisionmade based on what will provide the best chance for viability of thecells, and the best opportunity for their continued development intocells that can function in the scaffold in vivo in order to signal andpromote tissue regeneration.

The following list includes some of the cells that may be used asadditional cellular components of the composition of the invention: ahuman embryonic stem cell, a fetal cardiomyocyte, a myofibroblast, amesenchymal stem cell, an autotransplanted expanded cardiomyocyte, anadipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, amyoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, anembryonic stem cell, a parenchymal cell, an epithelial cell, anendothelial cell, a mesothelial cell, a fibroblast, a myofibroblast, anosteoblast, a chondrocyte, an exogenous cell, an endogenous cell, a stemcell, a hematopoetic stem cell, a pluripotent stem cell, a bonemarrow-derived progenitor cell, a progenitor cell, a myocardial cell, askeletal cell, a fetal cell, an embryonic cell, an undifferentiatedcell, a multi-potent progenitor cell, a unipotent progenitor cell, amonocyte, a cardiomyocyte, a cardiac myoblast, a skeletal myoblast, amacrophage, a capillary endothelial cell, a xenogenic cell, an allogeniccell, an adult stem cell, and a post-natal stem cell.

In particular, human embryonic stem cells, fetal cardiomyoctes,mesenchymal stem cells, adipocytes, bone marrow progenitor cells,embryonic stem cells, adult stem cells, or post-natal stem cellstogether with growth factors or alone with matrix scaffold optimizemyocardium regeneration in vivo.

Cells can be seeded directly onto matrix scaffold sheets underconditions conducive to eukaryotic cell proliferation. The highly porousnature of extracellular matrices in particular will allow diffusion ofcell nutrients throughout the membrane matrix. Thus, cells can becultured on or within the matrix scaffold itself. With the emulsifiedextracellular matrix compositions, or with some of the otherformulations, the cells can be co-cultured with the extracellular matrixmaterial before administration of the complete composition to thepatient.

Additional Components: Peptides, Polypeptides, or Proteins

In addition to a native ECM scaffold, or a synthetic scaffold, or amixture of the two, peptides, polypeptides or proteins can be added.Such components include extracellular structural and functional proteinsin admixture so as to mimic either heart ECM, or other native ECMs thatare capable of regenerating at least some reasonable percentage of thedefective myocardium, for example at least 30%, preferably more than50%. Effective regeneration of the myocardium relies on theextracellular matrix scaffold by its structure and components. Mimickingthe native explant material as closely as possible thus optimizes theopportunity for regeneration using a composition comprising some nativeECM, albeit treated, but also with additional components.

The peptides, polypeptides or proteins that can be added to the scaffoldare: a collagen, a proteoglycan, a glycosaminoglycan (GAG) chain, aglycoprotein, a growth factor, a cytokine, a cell-surface associatedprotein, a cell adhesion molecule (CAM), an angiogenic growth factor, anendothelial ligand, a matrikine, a matrix metalloprotease, a cadherin,an immunoglobin, a fibril collagen, a non-fibrillar collagen, a basementmembrane collagen, a multiplexin, a small-leucine rich proteoglycan,decorin, biglycan, a fibromodulin, keratocan, lumican, epiphycan, aheparan sulfate proteoglycan, perlecan, agrin, testican, syndecan,glypican, serglycin, selectin, a lectican, aggrecan, versican, nuerocan,brevican, cytoplasmic domain-44 (CD-44), macrophage stimulating factor,amyloid precursor protein, heparin, chondroitin sulfate B (dermatansulfate), chondroitin sulfate A, heparan sulfate, hyaluronic acid,fibronectin (Fn), tenascin, elastin, fibrillin, laminin,nidogen/entactin, fibulin I, fibulin II, integrin, a transmembranemolecule, platelet derived growth factor (PDGF), epidermal growth factor(EGF), transforming growth factor alpha (TGF-alpha), transforming growthfactor beta (TGF-beta), fibroblast growth factor-2 (FGF-2) (also calledbasic fibroblast growth factor (bFGF)), thrombospondin, osteopontin,angiotensin converting enzyme (ACE), and vascular epithelial growthfactor (VEGF).

Typically, the additional peptide, polypeptide, or protein componentwill comprise an amount of the composition by weight selected from thegroup consisting of greater than 0.1%, greater than 0.5%, greater than1%, greater than 1.5%, greater than 2%, greater than 4%, greater than5%, greater than 10%, greater than 12%, greater than 15%, and greaterthan 20%.

Whether a particular protein component or combination of components iseffective for myocardial tissue regeneration can be tested by contactingthe composition with defective myocardium in a test animal, for examplea dog, pig, or sheep, or other common test mammal. Myocardial tissueregeneration and myocardium contractility are both indicia to measurethe success of the composition and procedure, by procedures standard inthe art. In addition, a small sampling of the regenerated tissue can bemade to determine that new extracellular matrix and new tissue has beenmade. As to what balance between structural extracellular matrixproteins and functional ones to use in a given composition, natureprovides direction. Most ECMs are predominantly made up of structuralproteins by dry weight. Thus only a small portion of functional proteinsby weight are needed for effective myocardial tissue regeneration.

Peptides, polypeptides or proteins for the composition may be formulatedas is standard in the art for the particular class of protein, and thatformulation may be added to the extracellular matrix material (ofwhatever formulation) for delivery into the patient.

Alternatively, the protein molecules may be covalently linked to anappropriate matrix molecule of any of the matrix formulations. Covalentlinking of the protein molecules to molecules of the matrix may beaccomplished by standard covalent linking methods known in the art.

Additional Components: Vector Expressing DNA, Nutrients, Drug Molecules

Some of the proteins required for the composition can be geneticallysynthesized in vivo with DNA and vector constituents. Thus a vectorhaving a DNA capable of targeted expression of a selected gene cancontribute a bioactive peptide, polypeptide, or protein to thecomposition. Standard in vivo vector gene expression can be employed.

In addition, other additives such as a nutrient, a sugar, a fat, alipid, an amino acid, a nucleic acid, a ribo-nucleic acid, may providesupport to the regenerative process in vivo in the composition. Finally,also a drug, such as a heart regenerating or angiogenesis promoting drugmay be also added to the composition, in such a form as, for example, anorganic molecule, an inorganic molecule, a small molecule, a drug, orany other drug-like bioactive molecule.

Formulations

A formulation of extracellular matrix material can be an emulsified orinjectable material derived from mammalian or synthetic sources. Theextracellular matrix material can be emulsified or made into aninjectable formulation by standard procedures in the art, and maintainedas an emulsion or injectable until delivered to the patient. Oncedelivered to the patient, an environment is established (by some changesuch as a change in pH) so that the extracellular matrix molecules (bethey mammalian or synthetic) polymerize to form a matrix scaffold.

Depending on the nature of the scaffold selected, and depending on whichadditional components are used, the scaffold component and theadditional component can be formulated together in the same way, or indifferent ways that are however but delivery-compatible with each otherfor delivery purposes. Options for formulation of the scaffold include asolid sheet, multilaminate sheets, a gel, an emulsion, an injectablesolution, a fluid, a paste, a powder, a plug, a strand, a suture, acoil, a cylinder, a weave, a strip, a spray, a vapor, a patch, a sponge,a cream, a coating, a lyophilized material, or a vacuum pressedmaterial, all of which are standard in the art.

Formulation of the additional components, when they are notscaffold-like is generally accomplished using some form of aninjectable, semi-gel, or emulsified material, although powdered formsmay also then be combined with a hydration-promoting solution atdelivery. Thus, formulations for the additional components willgenerally comprise formulations of the nature of a gel, an emulsion, aninjectable solution, a fluid, a paste, a spray, a vapor, a cream, and acoating. Dried materials that are hydrated either at delivery or justbefore delivery are powders, such as lyophilized materials.

Cells can be added in from a culture, or can be co-cultured with thematrix component of the composition. Proteins can be added into thecomposition, or covalently linked to matrix molecules. DNA can be addedin with their vectors for expressing proteins in vivo. Other additivescan be combined with the matrix component as is practical for thedelivery of the composition (for example, as an injectable or acomposition administered with a percutaneous catheter) and as ispractical for maintaining bioactivity of the molecules or components invivo.

Fluidized forms of native extracellular matrices are described, e.g. inU.S. Pat. No. 5,275,826. The conminuted fluidized tissue can besolubilized by enzymatic digestion including the use of proteases, suchas trypsin or pepsin, or other appropriate enzymes such as a collagenaseor a glycosaminoglycanase, or the use of a mixture of enzymes, for aperiod of time sufficient to solubilize said tissue and form asubstantially homogeneous solution.

The present invention also contemplates the use of powder forms ofextracellular matrix scaffolds. In one embodiment a powder form isprepared by pulverizing basement membrane submucosa tissue under liquidnitrogen to produce particles ranging in size from 0.1 to 1 mm.sup.2.The particulate composition is then lyophilized overnight and sterilizedto form a solid substantially anhydrous particulate composite.Alternatively, a powder form of basement membrane can be formed fromfluidized basement membranes by drying the suspensions or solutions ofcomminuted basement membrane. The dehydrated forms have been rehydratedand used as cell culture substrates without any apparent loss of theirability to support cell growth.

Delivery Modes

The mode used for delivery of the compositions of the invention to thedefective myocardium may be critical in establishing tissue regenerationin vivo. Standard delivery to myocardial sites can be used forinjectable, fluidized, emulsified, gelled, or otherwise semi-fluidmaterials, such as direct injecting (e.g. with a needle and syringe), orinjecting with a percutaneous catheter. For materials that have beenrendered wholly or partially vaporized, force-driven delivery of thematerial can be used, for example, CO₂ powering emission of fineemulsion, micronizing an injectable solution, ink jet delivery, spraywith a conventional atomizer or spray unit, or other type of vaporizeddelivery. Some of these vaporized formulations can be delivered using apercutaneous catheter adapted for delivery of a vaporized formulation.

For materials that are essentially solid, such as some of the native orsynthetic scaffolds, physically depositing the material will be the mostprudent mode of delivery. For example a patch, sponge, strip, weave, orother geometrically defined material form should be placed at the siteof deposit either during surgery, or with a percutaneous minimallyinvasive catheter capable of depositing all or portions of solidmaterial at the site. Preferred modes of delivery will be minimallyinvasive delivery procedures, which reduce the risk of infection andprovide an easier recovery for the patient.

Where the scaffold component is in a different material form than theadditional components, care must be taken to orchestrate an effectivedelivery of both components to the site. For example, where the scaffoldis a solid sheet, and cells have been cultured and proteins hydrolyzed,the cells and proteins may be added to the scaffold prior to deliveryand the composition is then delivered in surgery. Alternatively, also insurgery, the solid sheet of scaffold may be delivered and the emulsifiedagents deposited on the sheet before closure. Where both the scaffoldcomponent and the additional components can be emulsified, with completeretention of functionality, the composition can be delivered together bydirect injection or percutaneous catheter delivery.

In all cases, before a mode is used to treat a patient, the feasibilityand effectiveness of any one delivery mode or combination of modes canbe tested in a test mammal prior to actual use in humans.

Methods of Use

A site of defective myocardium is identified and the appropriatecomposition of a scaffold component and additional components is madeand formulated. The formulated composition is delivered by anappropriate means to the site of defect. The site and mammal areobserved and tested for regeneration of the defective myocardium todetermine that an effective amount of the composition has beendelivered, particularly to observe new tissue growth, and also todetermine that the new tissue has the contractility necessary for it tofunction usefully as myocardium. Tissue growth and contractility can betested and observed by standard means, for example as described inBadylak et al, The Heart Surgery Forum, Extracellular Matrix forMyocardial Repair 6(2) E20-E26 (2003).

Goals for contractility in the defective myocardium include observed andmeasured contractility in an amount measured against contractility of anormal heart selected from the group consisting of greater than 10%,greater than 20%, greater than 30%, greater than 40%, greater than 50%,greater than 60%, greater than 70%, greater than 80%, greater than 90%,and greater than 95% of normal myocardial contractility in vivo.

The method step of contacting the defective myocardium or site of absentmyocardium with a composition of the invention can be accomplished bymeans discussed in the delivery section, including, delivering thecomposition by injecting, suturing, stapling, injecting with apercutaneous catheter, CO₂ powering emission of fine emulsion,micronizing an injectable solution, inkjet delivery, physicallydepositing a sponge, physically depositing a patch, physical depositinga strip, or physically depositing a formed scaffold of any shape.

A complementary method of use of the compositions of the inventioninclude a method of inducing angiogenesis in myocardium at a site ofischemia by similarly contacting said ischemic myocardium with acomposition of the invention in an amount effective to induceangiogenesis in the myocardium at the site of ischemia. Effectivenesscan be measured by measuring vascularization at the site, using standardbiomedical procedures for such analysis.

EXAMPLES Example 1

An emulsion of urinary bladder submucosa (UBS) is prepared usingstandard emulsifying techniques. The emulsion is free of endogenouscells. This preparation is maintained as an emulsion by controlling thepH during storage of the emulsion before it is admininstered to thepatient. In a minimally invasive procedure, a percutaneous catheterdevice is loaded with sufficient quantity of the emulsified UBS toaddress a defect in a human heart, the defect having been identifiedpreviously by imaging. The catheter is directed to the site of themyocardium in need of tissue regeneration using sonographic orradiographic imaging. Upon contact with the site, the emulsion isreleased and the catheter is withdrawn. The tissue regeneration processis monitored by sonography for several weeks or months post-delivery ofthe emulsion.

Example 2

An emulsion of decellularized immunogenic liver basement membrane (LBM)is prepared using standard known techniques. While maintaining theemulsion state of the LBM, adult stem cells are co-cultured with theemulsion using standard stem cell culturing techniques. When the cellsare ready, the entire composition is loaded into a catheter forpercutaneous delivery to a human patient in need of tissue regenerationat a site of defective or absent myocardium. The emulsion with theco-cultured cells is delivered to the patient: a percutaneous catheteris loaded with the emulsion and directed to the site of the myocardiumin need of tissue regeneration using sonographic or radiographicimaging. Upon contact with the site, the emulsion is released and thecatheter is withdrawn. The tissue regeneration process is monitored bysonography for several weeks or months post-delivery of the emulsion.

Example 3

An injectable emulsion of decellularized immunogenic stomach submucosa(SS) is prepared using standard known techniques. An aliquot ofglycoaminoglycan (GAG) protein is covalently linked to some of themolecules of the matrix emulsion using standard covalent linkingprocedures for proteins. While maintaining the emulsive state of the SS,bone marrow progenitor cells are co-cultured with the emulsion usingstandard progenitor cell culturing techniques. An aliquot oftransforming growth factor protein is added to the co-culturingcomposition before delivery to the human in need of myocardial tissueregeneration. The emulsion complete with cells and proteins is loadedinto a percutaneous catheter which is directed to the site of themyocardium in need of tissue regeneration using sonographic orradiographic imaging. Upon contact with the site, the emulsion isreleased and the catheter is withdrawn. The tissue regeneration processis monitored by sonography for several weeks or months post-delivery ofthe emulsion.

All references cited are incorporated in their entirety. Although theforegoing invention has been described in detail for purposes of clarityof understanding, it will be obvious that certain modifications may bepracticed within the scope of the appended claims.

1. A method of making a composition comprising at least a portion of anextracellular matrix scaffold component from a mammalian source and afirst additional component selected from the group consisting of acollagen Present in myocardial tissue, a collagen type IV protein, acollagen type VIII protein, a collagen type X protein, a multiplexin, amatrix heparin sulfate protoeglycan, a laminin, a nidogen, an agin, anda perlecan, and a second additional component selected from the groupsconsisting of: a) a plurality of synthetic extracellular matrix-likescaffold-forming molecules b) a cell, c) a peptide, polypeptide, orprotein, and d) a vector having a DNA capable of targeted expression ofa selected gene, and e) a nutrient, a sugar, a fat, a lipid, an aminoacid, a nucleic acid, a ribo-nucleic acid, an organic molecule, aninorganic molecule, a small molecule, a drug, or a bioactive molecule,said method comprising contacting the extracellular matrix scaffoldcomponent with at least one first additional component and at least onesecond additional component.
 2. The method of claim 1, wherein saidextracellular matrix scaffold component is formulated in a material formselected from the group consisting of a solid sheet, multilaminatesheets, a gel, an emulsion, an injectable solution, a fluid, a paste, apowder, a plug, a strand, a suture, a coil, a cylinder, a weave, astrip, a spray, a vapor, a patch, a sponge, a cream, a coating, alyophilized material, and a vacuum-pressed material.
 3. The method ofclaim 1, wherein said first or second additional component is formulatedin a material form selected from the group consisting of a gel, anemulsion, an injectable solution, a fluid, a paste, a powder, a plug, astrand, a spray, a vapor, a cream, a coating, a lyophilized material anda vacuum-pressed material.
 4. The method of claim 1, wherein theextracellular matrix scaffold component is from a source selected fromthe group consisting of small intestine submucosa (SIS), urinary bladdersubmucosa (UBS), urinary basement membrane (UBM), liver basementmembrane (LBM), stomach submucosa (SS), collagen from animal sources,collagen from plant sources, synthesized extracellular matrix incultures from cells (Matrigel™), dermal extracellular matrix material,subcutaneous extracellular matrix material, large intestineextracellular matrix material, placental extracellular matrix material,ornamentum extracellular matrix material, heart extracellular matrixmaterial, and lung extracellular matrix material.
 5. The method of claim1, wherein the second additional component is a plurality of syntheticextracellular matrix-like scaffold-forming molecules, and said syntheticmolecules are selected from the group consisting of polyethyleneterephthalate fiber (Dacron), polytetrafluoroethylene (PTFE),glutaraldehyde-cross linked pericardium, polylactate (PLA), polyglycol(PGA), hyaluronic acid, polyethylene glycol (PEG), polyethelene, anucleic acid polymer, nitinol, and collagen from non-animal sources.